Process for the preparation of therapeutic compositions from spleen tissue



Patented Dec. 8, 1953 "PROCESS FOR- THE PREPARATION F THERAPEUTIC COMPOSITIONS -=FROM Jesus MilAmozurrutia, MexicorCity, Mexico, as-

.signor to Mirantla Brothers Trading .Corporas- "tion, New York, "N. Y., acorporation of New *York N0 Drawing. Original application November 2 7,

1945, Serial No. 631,230, new Patent ,N 0.. 2,628,961, dated Februaryllfl, 1953. ,Dividedl' and this :application J alil ary ,28; 1953; Serial 2 Claims. (01. 167-44) ".This--.:application is ;;a division. of the', copen d-. mgapplication, SeriaLN 0.; 631,230,;filed-Novembera2i1n1945.

inventionrelates. to; therapeutic compositions, andmore partie-ularlyto compositions-canahle .of removing or diminishing the manifestations'of theallergic-stateand adapted for the treatment of. certain. other .ailments.

In. 1902 ,Charles Richet: found that oneinoculation aofea given protein tends to, render an animore. sensitive. instead of becoming immune to that.;.-p-r0.tein. 'IIo-this phenomenon of hypersensitivenesswinexperimental animals, he gave the name anaphylaxis. .Von-Pirquet in; 1-906 employed.:the ,term fal1ergy .to. denote a similar kind -of lsensitivi tyin humanv beings. By follow- :ing-thistline of thought..:inye stigators cameto the-,conclusionthat there-were some diseases in human beings which:ar.e characterized by an altered. reactivity .to external agents which. result in, :for. example,- hay. fever, asthma, and eczema. The .term al1ergy..-is .now appliedto; this group of diseases. I 7

.There. i still marked uncertainty among investigatorswith respect to the exact limits of a1- lelgy. .Thismncertainty arises from the observa- 'tionfthaauinl-spite.of all known .factors concern- :iiig, the;.mechanism .of allerg there are other components .which are. still unknown. In-view of thissituation, Doernin 1912 1-(Doerr, .R.,.Allergic Bhenomena in .Hand. dl'-NOIII1."11. -Path., Physiol, 131.659,,1929) choseallergy as a termto include a-lLabnormaLand specific .react;ions; of, the body to. .foreign, ordinarily innocuos, substances; Rackemann Vaughan. Schick and Rowe, among others,..r.ecentlyreafiirmedsuch use of the term allergy. :Since: nearly every chronic or acute disease shows varying degreesiof allergicreactions, schicklcoined the term fhypererey to designate outstanding allergic manifestations, :such: as .hay

feyer,.-asthma .andeczema.

The diagnosis of a given disease. aslsa'llergic and the identification of the causative antigen in the methods heretofore'employed for the treatmentotallergyprecedesxthe management ofthe allergic/patient. J According to; Rowe cthelrcutine laboratory studies shouldincludei: -.A complete blood zcountnurinalysiaa complement'fixation-or flocculation test, and roentgen-ray studiesof the sinuses: chest, 'gastro-intestinal. tract, including, ifrindications exist; themolonand gall bladder; lna-additionwhe suggestseas aids in difierenti'al diagnosis, 2 estimation 320i. eosinophiles in- -the bleach- 1nasalwsecretionsc:or; oral secretions; 1 gastzgic or mucousmnd gastrieanalysis,-preferably-of thefunctional type, with the alcohol mealor histamineinjection in all gastro-intes-v tinal disordersto cover the. occassional-occurrence-of achylia orhypoclarhydria. Other tests aressuggested, all of which involve considerable time. to I diagnose properlyv the allergic. conditions andindicate a propencourse. of treatment. Urifortunately, these clinicalstests are by no? means without danger. Moreover, skin tests are not always reliable and are-nott uniformly accurate inthe hands of difterentworkers.

The treatment of an allergiccondition is..as.a rule a painstaking and prolonged-procedure. According to Urbach, there-are fiveinethods of combating allergic diseases. -The fivemethods of: treatment 1 are Prophylaxis Specific hyposensitiaation Heterospecific hyposensitization Deallergization Symptomatictherany ma t-owe:

allergic2disturbances in need of treatment. -Ae

cording .to Rowe; whenthe-occurrence ofall 0hvious manifestations of nasal-bronchial, cutaneous,..gastro -intestinal, ocular, -genito-urina-ry --a*l'- IQIgYg-Elldalltirgic migraine I is accurately recogize'd, however, :the incident of active allergic manifestations. of.:either a= maj'oro m-inor degree probablyilies.betWeenQZBQand Theoccurrence of allergy would immediately be-raised close-to 11:001% Yif-disturbances, mild orseverei arising .from. bacterial allergy,--Whieh is so; important inisome infections-were included. It is'obvious, therefore, that therapeutic procedures are inadequate. for .the treatment of allergic-conditions. Earmerisoftheopinion-thatthere is a factor which is-common -toall allergicconditionsQ He also believes that 1 a single -treatment imight he found for the t'reatmentoi all of-th s -conditions, but heretofore no successful single treatment has been proposed.

In accordance with this invention, the symptoms of the allergic state may disappear or diminish substantially by the administration of the new and novel therapeutic compositions of this invention. These compositions comprise haptenes or poly heterophilic substances which play the role of polyvalent antigens. They are derived from various organs and body tissues of mammals and are substantially insoluble in benzol, toluene, acetone, and cold ethyl alcohol (e. g. 16-20 C.) and are soluble in water and hot ethyl alcohol. They are glycosides and when treated with weak acids, they partially hydrolyze. When administered into mammals they produce, at the site of injection, local eosinophilic reactions. These substances when administered are free from fats and proteins.

The compositions of this invention are prepared from substantially all organs and tissues, such as the spleen and muscle of mammals, by means of various solvents, in which the compositions are soluble and the other constituents of the tissue, such as fats and lipoids, are insoluble, or alternately by means of solvents in which the compositions are insoluble and the other constituents of the tissue are soluble. For example, the spleen of a mammal, such as that of a hog, which contains these compositions is first extracted with benzol in which the fats of the spleen are substantially soluble. The resulting residue is then treated with a solvent, such as water, in which the compositions are soluble, and

:the resulting water solution is purified by any suitable means to remove the proteins therefrom. .Alternately, the resulting residue is treated with a solvent, such as ethyl ether, and the mixture :filtered, the residue being discarded. The compositions of this invention may be obtained from the filtrate by first evaporating it and dissolving the resulting residue in water, at a temperature of about 16 to 20 C. :aqueous solution of the resulting composition is placed in suitable containers, such as ampules, and sterilized by any convenient means, such as .heating to a temperature of about 120 C. for a period of 20 minutes to one hour.

In either procedure, an

The compositions of this invention are glyco- :sides and give the characteristic reaction for 1glycosides. When .001 milligram of the compositions of this invention are injected into mam- :mals, such injections produce a local eosinophilic :reaction which is readily discernible by a micro- ,scopic examination of a smear taken from the zsite of injection after a period of from 3 to 5 hours. of this invention are administered in a water Desirably, the therapeutic compositions .solution intramuscularly. In certain cases of gastro-intestinal allergies, the therapeutic compositions may be administered orally. The com- 4 compositions are administered for a period of about 6 days, after which there is no further treatment for a period of from one to several weeks. The therapeutic compositions are desirably not again administered until allergic symptoms are again manifested by the patient.

A more comprehensive understanding of this invention is obtained by reference to the following examples. The proportions and various solvents employed in these examples are merely illustrative and are not to be construed as limitations on the process of preparing the compositions of this invention.

Example 1 Ten kilograms of fresh hog spleen are ground in any suitable mechanism, such as a meat grinder, and then dried in the air, preferably employing air by means of circulating fans for evaporating the water therefrom. Subsequently the dried product is ground by any suitable means, such as a grain mill, to a finely divided state. To the resulting powder is added about 5 kilograms of pure benzol. The mixture is agitated vigorously and permitted to stand for a period of about 24 hours. The mixture is then filtered, and to the residue is added 5 kilograms of benzol. This benzol mixture is agitated vigorously for a few minutes, permitted to stand for a period of 24 hours and filtered. The residue is then spread out into a very thin layer and dried, desirably on filter paper or glass. The residue is then extracted with absolute ethyl ether. About 2 kilograms of ether are employed for this purpose. The compositions of this invention are soluble in the ethyl ether, while the proteins and other undesired materials are insoluble. The mixture is then filtered, and the residue discarded. The filtrate is evaporated, and to the resulting residue is added 1 liter of distilled water. The mixture is agitated with a glass rod or other agitating device that does not contaminate the resulting material. Extraction with water is conducted at a temperature between about 16 to 20 C. The mixture is then filtered and the residue discarded. The filtrate is placed in ampules or other suitable containers for administration. Desirably, the material after being placed in ampules is sterilized by the usual conventional methods, such as heating to about C. in an autoclave for a period of 20 minutes to 1 hour. When a water solution containing .001 milligram of the composition prepared in accordance with this example is injected intramuscularly into mammals, and a smear of the tissue at the site of injection three to five hours after injection is microscopically examined an eosinophilic reaction is observed. Standard tests confirm that the composition prepared in accordance with Example 1 is a glycoside and that the final water solution of that composition is substantially free of fats and proteins.

Example 2 Ten kilograms of fresh hog spleen are ground and extracted with benzol as in Example 1 to separate the fat constituents from the compositions of this invention. To the residue obtainedfrom this step is added about 500 cc. of distilled water. The mixture is agitated, permitted to stand for about 3 hours and then filtered. To the filtrate is added a sufiicient quantity of cold absolute ethyl acohol (about 16 to 20 C.) to precipitate the material and the mixture is filtered. About 2 to 3 liters of absolute ethyl alcohol are required for this purpose. To the residue is again added about 250 cc. of water. The mixture is agitated, permitted to stand for about 3 hours and filtered. To the filtrate is added about 1 to 1 liters of cold absolute ethyl alcohol to reprecipitate the composition of this invention. It is again filtered and the residue dissolved in about 250 cc. of water. Suflicient alcohol is then added to produce a mixture containing 80 to 85% alcohol and the mixture heated to the boiling point of the alcohol. The compositions of this invention are soluble in ethyl alcohol heated to this temperature. The mixture is filtered while the mixture is at the elevated temperature, and the filtrate permitted to cool to room temperature or lower. Desirably, the pH of the mixture is adjusted to between 5 and '7 to assure a good yield of the precipitate. Since the therapeutic compositions of this invention are substantially insoluble in cold ethyl alcohol, it precipitates out under these conditions. The mixture is then filtered. If the precipitate indicates a contamination by other than a pure White color, the procedure of purification by dissolving in water and precipitating with alcohol is repeated. The precipitate is purified by recrystallization with ether. therapeutic. composition is injected into mammals, such as the guinea pig, a local eosinophilic reaction is observed by microscopic examination in smears taken from the mammal at the sight of the injection three or four hours after the injection. The resulting composition gives no reaction for proteins or fats and a position reaction for glycosides.

Although in the specific examples the therapeutic compositions of this invention are derived from the spleen of a mammal, such compositions may be obtained from other body tissues of the mammals, by following the procedure outlined in the examples. Whether derived from the spleen or other tissue of mammals, the compositions are substantially the same, and .001 milligram of such compositions produce a local eosinophilic reaction when injected into mammals.

When .001 milligram of the resulting The terms and expressions which I have employed are used as terms of description and not of limitation, and I have no intention, in the use of such terms and expressions, of excluding any equivalents of the features shown and described or portions thereof, but recognize that various modifications are possible within the scope of the invention claimed.

What is claimed is:

1. The method of preparing a therapeutic composition comprising separating the benzol soluble from the benzol insoluble portions of the spleen tissue of a mammal, dissolving the benzol insoluble portion in water, separating any undissolved water insoluble materials therefrom, adding suificient cold ethyl alcohol to the water extract to produce a concentration of to ethyl alcohol, separating the precipitate formed, redissolving said precipitate in water, removing any water insoluble material, adding sufficient alcohol to produce a mixture containing 80 to 85 ethyl alcohol, heating said mixture to about the boiling point thereof, removing any insoluble materials in said mixture, adjusting the pH of said mixture between 5 and 7 and permitting it to cool, removing the resulting precipitate and recrystallizing said precipitate with ethyl ether.

2. The method of producing a therapeutic composition which comprises separating the benzol soluble from the benzol insoluble portions of the spleen tissue of a mammal, extracting with water the water soluble components of said benzol insoluble portion, separating any insoluble matter from the resulting extract, adding cold ethyl alcohol tothe water extract to precipitate the therapeutic composition, forming a hot ethyl alcohol extract of the precipitate, separating the insoluble matter therefrom and recovering the components of the resulting extract which are insoluble in cold ethyl alcohol.

' JESUS M. AMOZURRUTIA.

No references cited. 

1. THE METHOD OF PREPARING A THERAPEUTIC COMPOSITION COMPRISING SEPARATING THE BENZOL SOLLUBLE FROM THE BENZOL INSOLUBLE PORTIONS OF THE SPLEEN TISSUE OF A MAMMAL, DISSOLVING THE BENZOL INSOLUBLE PORTION IN WATER, SEPARATING ANY UNDISSOLVED WATER INSOLUBLE MATERIALS THEREFROM, ADDING SUFFICIENT COLD ETHYL ALCOHOL TO THE WATER EXTRACT TO PRODUCE A CONCENTRATION OF 80 TO 85% ETHYL ALCOHOL, SEPARATING THE PRECIPITATE FORMED, REDISSOLVING SAID PRECIPITATE IN WATER REMOVING ANY WATER INSOLUBLE MATERIAL, ADDING SUFFICIENT ALCOHOL TO PRODUCE A MIXTURE CONTAINING 80 TO 85% ETHYL ALCOHOL, HEATING SAID MIXTURE TO ABOUT THE BOILING POINT THEREOF, REMOVING ANY INSOLUBLE MATERIALS IN SAID MIXTURE, ADJUSTING THE PH OF SAID MIXTURE BETWEEN 5 AND 7 AND PERMITTING IT TO COOL, REMOVING THE RESULTING PRECIPITATE AND RECRYSTALLIZING AND PRECIPITATE WITH ETHYL ETHER. 